PROJECT SUMMARY ABSTRACT The vasoactive peptide adrenomedullin 2/intermedin (AM2/IMD) has important actions in human physiology and disease such as vasodilation, physiologic and pathologic angiogenesis as well as potent protective effects in the cardiovascular and renal systems. Actions of AM2/IMD have been attributed to activation of several signaling intermediates including cAMP and Ca2+ downstream of its G protein-coupled receptor, the calcitonin receptor-like receptor (CLR). Unfortunately, there is little mechanistic insight into how AM2/IMD binds and activates CLR. CLR pharmacology is complicated because it heterodimerizes with any one of three receptor activity-modifying proteins (RAMP1, -2, or -3) that modulate its response to AM2/IMD and the related vasoactive peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). How AM2/IMD, CGRP, and AM act through shared RAMP:CLR receptor complexes to promote their unique signaling outcomes remains unclear. This limits our understanding of how AM2/IMD elicits its broad range of actions in human physiology and hinders our ability to exploit AM2/IMD signaling for drug development. I will test the hypothesis that AM2/IMD adopts a unique receptor-bound conformation and that it promotes a pattern of biased G protein activation at RAMP:CLR complexes that is distinct from those of AM and CGRP. I will test this using rigorous biochemical, pharmacological, and structural methods in two aims: 1) Define the molecular basis for AM2/IMD recognition by soluble RAMP:CLR extracellular domain (ECD) complexes, and 2) Define the G protein-coupling preferences of each full-length receptor complex promoted by AM2/IMD as compared to CGRP and AM. For Aim 1 I purified each of the three ECD complexes as tethered RAMP ECD-CLR ECD fusion constructs and found that AM2/IMD exhibited binding preferences that were distinct from those of CGRP and AM. I solved a 2.05 resolution crystal structure that demonstrated a strikingly unique triple b-turn structure of AM2/IMD bound to the RAMP1-CLR ECD. I will determine an AM2/IMD-bound crystal structure of the RAMP3-CLR ECD to fully understand how AM2/IMD binds the different receptor ECDs, and provide crucial insights into how RAMP3 modulates CLR. For Aim 2 we determined conditions to co-express and solubilize the three full-length RAMP:CLR complexes, which form detergent-stable ligand-free complexes. This provides a unique opportunity to study how the three peptides promote coupling of different G-proteins. We will use a native-PAGE method to determine coupling preferences to unpurified receptor complexes and we will purify the ligand-free complexes to study G-protein coupling using a fluorescence anisotropy assay. These biochemical studies will be correlated with pharmacological studies of cAMP and Ca2+ signaling bias in human cell lines that express the RAMP1:CLR (SK-N-MC) or RAMP2:CLR (HUVEC). Successful completion of these aims will provide crucial insights into AM2/IMD function that will enable AM2/IMD-based drug development.!